DOUBLY-LABELLED WATER：doubly-labelled水.doc

25DOUBLY-LABELLED WATERCALCULATION PROGRAMUSER GUIDE(version 1.0)J.R. SPEAKMANAndC. LEMENFunded by UK NERC Scientific Services& The University of Aberdeen, Scotland UKJanuary 1999CONTENTS1.Background 32.Getting started 4-53.Estimating injectate enrichments24-254.Two sample experiments64.1 Entering new data4.2Dilution74.3Group74.4Background worksheet8-94.5Set Options104.6Initial worksheet10-124.7 Final worksheet134.8Save data144.9 Run analysis14-225. Multiple sample experiments6. Tritium7. Acknowledgements1.BackgroundThis program calculates the CO2 production and inferred daily energy expenditure and food intake from isotope enrichment data derived from doubly-labelled water studies. The program uses all the alternative equations that had been published by 1997. Details of the derivation of the equations along with a discussion of which equations are most appropriate under differing circumstances can be found in Speakman (1997) The program was written by C. Lemen of Natureware Inc, USAFunding for this project was provided by the United Kingdom Natural Environmental Research Council (NERC) Scientific Services and The University of Aberdeen, Scotland, UK. The program is provided free of charge to bona fide users of the DLW method without guarantees or liability. The only condition of using the program is that its use is acknowledged in any publication. This User Guide may serve as an appropriate citation. The program works in the windows environment of any PC. Minimum system requirements are a 486 machine with 8 Mb RAM running windows 3.12.Getting startedTo load the program transfer the DLW.exe file from the disc, or directly from the web site on to the hard drive of your PC. Do not locate it in a special folder. Create an icon, from which the program can be accessed directly, or use the Start and run options (command line dlw.exe) to start the program. When running correctly the program should open up a window with a standard task bar at the top of it and four pairs of icons below.Check the version number under Help and AboutThe version should be 1.0 or above.These pairs of icons allow direct access into the different operations that are available. Alternative access is via the File command on the task bar.2.1 Two sample studies The first pair of icons. These are protocols where the subject has been sampled for background, injected with isotope, sampled for an initial isotope enrichment and then released to be recaptured and sampled again at some later time. The first icon concerns new data.The second recovers saved data from a file. This second option only recovers data that has been saved previously using this program. Filenames of recovered files must have the form *.dl2Alternatively the same two options can be accessed by clicking on File on the task bar and selecting “New 2 sample” or “Open 2 sample”2.2 Multiple sample studies. The second pair of icons. These are protocols where the subject has been sampled repeatedly across several days with the intention of determining the elimination rates of the isotopes using regression. This does not refer to essentially two sample studies where it happened that the animal was recaptured twice. For such analyses the “Two sample protocol” should be used twice (but beware of lack independence of the derived estimates). Again there are two icons the first is for entering new data and the second is for recovering saved data from a file. This second option only recovers files that have been saved by the program and have the form *.dlmAlternatively these same two options can be accessed under File on the task bar and selecting “New multi-sample“ or “Open multi-sample“.2.3 Isotope enrichments in injectate solutionsThe third pair of icons. These are intended to be used with data derived from dilution experiments where known amounts of the injectate have been injected into known amounts of water at background enrichment. Again there are two icons the first is for entering new data and the second is for recovering saved data from a file. This second option only recovers files that have been saved by the program and have the form *.enrAlternatively these same two options can be accessed under File on the task bar and selecting “New estimate enrich“ or “Open enrichment“.2.4 Group propertiesThe last two icons. Some calculations of CO2 production using DLW depend on parameters derived from the local group of animals in a particular study. This means calculations must be performed using the two sample or multiple sample options, and the data appended to a group file for each individual. Once a group has been completed the local parameters can be derived and the calculations which depend on these local group parameters estimated by re-running the two or multiple sample options and specifying the group file name at the appropriate point (see below). These two icons allow manual creation of a group file, or inspection of a file that has already been created. Alternatively the same functions can be accessed under File on the task bar and selecting “New Group” or “Open group”3. ESTIMATING INJECTATE DILUTIONSThe module for estimating injectate enrichments is accessed either via the 3rd set of paired icons or from the task bar under file and choosing either new estimate enrich or open enrichmentNEW ESTIMATES OF ENRICHMENTThis module allows estimation of the enrichment of an injectate solution using a convergence iterative algorithm. The algorithm starts with estimates of the enrichments of both isotopes to set the molecular weights and then iterates the calculation using the derived enrichments to generate new molecular weights. The program performs 30 iterations. Convergence on a reasonable estimate normally occurs before 4 iterations are completed. The window consists of 11 boxes for entry of data derived from an injectate dilution experiment. The program can operate with enrichment data either in ppm or in delta. In the case of delta it is necessary to include the known isotope ratios for the reference compounds to which delta refers. The default data included in the windows when it is first opened are the reference ratios known for SMOW.On the line following these ratios are the estimated enrichments of deuterium and oxygen-18 in the injectate. These can be the manufacturers stated enrichment estimates or any other guesses of the likely enrichments in the solutions. Closer estimates minimise time to convergence but as the time is generally not an issue any values can be entered here. If a completely unknown solution is analysed the recommended values to use are 50 and 50.The next three boxes require weights for the vessel into which the background water and injectate were mixed. The first box requires a mass for the vessel alone. The next box for the vessel plus the background water, and finally the vessel plus the background water plus the injectate. The boxes are designed like this because this is typically the procedure followed in a dilution experiment and it obviates the need for the user to manually calculate how much background water and injectate are involved in the mix. Masses should be entered to four decimal places.The buttons above the following 4 boxes specify if the following data are in delta or ppm. Clicking on the buttons switches between the two types of unit. If ppm is selected the ratio at the top of the sheet becomes irrelevant. It is not possible to mix the data units. All enrichments must either be in delta or ppm. The four boxes require enrichment estimates for both isotopes in first the background water that is used as a diluent. These boxes are labelled Water 2H and water 18Oand second the mix of diluent and injectate which are labelled Water plus injectate 2H and Water plus injectate 18OOnce the data entry is completed there are 3 options.SAVE will save the data in the boxes into a file appended *.enrThese files can be reloaded into the worksheet using the open enrichment command under file or the second enrichment icon on the task bar. Cancel leaves the worksheet unalteredAnalyse performs an estimation of the enrichments of deuterium and oxygen-18 in the injectate solution. Analysis time is generally instantaneous. The output from analyse consists of six lines of data output. A typical example followsEnrichment of injectate2H ppm in water= 155.418O ppm in water= 2005.52H ppm in water plus injectate= 1999.018O ppm in water plus injectate= 6003.1ppm 2H enrichment in injectate= 130543ppm 18O enrichment in injectate= 284734The first four lines represent the mean values derived from the four input boxes.The final two lines represent the iterated calculation of enrichment of the injectate to the nearest ppm. If the input data are delta units then the output data are also in delta units.NOTE : the output program has a bug here and lists the output enrichments as ppm irrespective of whether the input is delta or ppm. The file can be printed to the default device printer on the PC by selecting PRINTAlternatively the data can be saved to a file using SAVE.(Note in version 1.0 this capability is disabled)Alternatively data can be entered into the window from an existing file by selecting the OPEN ENRICHMENT option under file or the second icon of the pairing on the task bar. (Note : there is a bug in the module which is used to re-open these files. If activated the module opens a window which is headed “Save as” and says the file type is “dilution” rather than enrichment. However, files listed in the window will be previously saved *.enr files and they should open correctly).4. TWO SAMPLE EXPERIMENTS4.1 Entering new dataOpening this option reveals a window labelled IDD-t2main. This window contains 5 boxes in which information can be entered plus 8 buttons which access further data entry screens.The 5 boxes are intended to contain information on the subject and the experiment it was involved in and are labelled :BoxMax inputSpecies33 charactersID #14 charactersLocation76 charactersDate10 charactersNotes3 lines x 46 characters on each Any information typed into these boxes will appear on the header of the output from the main calculation (section 4.9) under the appropriate heading. None of these boxes are compulsory fields. It is possible to make calculations with all the boxes empty. None of the information entered in these fields (e.g. date) is used in the calculations.4.2 DilutionThe dilution data entry button opens a small window where it is necessary to enter the enrichments of the two isotopes in the injectate solution. There are two boxes labelled hydrogen2 ppm and Oxygen18 ppm. These fields must contain the ppm enrichments of the injectate for the program calculations to continue. Data can be entered in 3 ways/1) Manually into the boxes. 2) The output from the injectate enrichment module can be entered directly into these fields by accessing the appropriate *.enr file. Clicking on the button “Open dilution file” allows access to the file searcher which will seek any files appended *.enr. (Note : not possible in version 1.0). 3) The output from a *.dln file that was saved from the dilution worksheet of a previous animal can be loaded directly into the boxes following the same procedures as detailed in 2).The second button “Save dilution file” allows manually entered data in these fields to be saved to a *.dln file for future reference. Cancel closes the window without saving the current settings in the fields. OK accepts the most recent values entered into the fields. Both fields are mandatory for the calculation to proceed. The enrichment must be entered as ppm. Other units of enrichment (e.g. delta SMOW-SLAP) are not acceptable.4.3 GROUPThe group box allows specification of a local group from which parameters can be derived for some of the DLW calculations. Clicking on GROUP opens a window in which an analysis is performed of the characteristics of any particular group of data. Within this window there is a button called Select group search window for files with the append *.grp. Selecting the group performs an analysis of the data held in the group file of the relationship between the dilution space and body mass of animals in the group. Four analyses are performed using the hydrogen and oxygen data for both plateau and intercept dilution spaces. A plot shows the relationships derived. Plateau data are in green, intercept data in red, oxygen symbols o and hydrogen symbols +.The regression parameters for these four plots are located in a table to the left of the plot. These show the intercept, gradient and r2 for each relationship. By inspecting these plots one can decide if using these group relationships might enhance the predictions of the final body water space from mass. Generally this will be useful if the r2 of the relationships are good ( 0.8). Clicking on the button above the table allows the selection or not of using these group data to derive the final dilution spaces.Once selected the group name appears in the box adjacent to the button.4.4 BACKGROUND WORKSHEETClicking on background worksheet opens a window which requests information on the background enrichments of the isotopes.There are four data fields within this window, all of which must be completed for the program to run. Data is entered in each field by clicking the mouse in that field to activate a cursorDateFormat for the date is mm/dd/yyyyThe program automatically inserts the /It is not possible to reverse past the /If you make a mistake then you must enter the whole field again or relocate the cursor using the mouse and change the bit that is wrong.To delete the whole field highlight the whole field using the mouse and delete using the delete button.The program will issue a warning if an invalid date has been entered (e.g. the 21st month etc) and progress will not be allowed to continue until this is rectified. It works for the year 2000. TimeTimes are entered on the 24 clock in the format hh:mmThe program will automatically enter the :The program will warn if an invalid time has been entered.If you make a mistake then you must enter the whole field again or relocate the cursor using the mouse and change the bit that is wrong.Hydrogen ppmThis box allows entry of background enrichment data for the hydrogen isotope. Up to 84 separate determinations of background hydrogen enrichment can be entered. Each enrichment must be entered on a separate line.Use the enter key to advance to the next lineImportant NOTE : after entering the last data item DO NOT PRESS ENTERIf the cursor moves on to the next line the program will assume the last data point is a 0. Oxygen ppmThis box allows entry of background enrichment data for the oxygen isotope. As with the hydrogen isotope up to 84 separate determinations of background oxygen enrichment can be entered. Each enrichment must be entered on a separate line.Use the enter key to advance to the next lineImportant NOTE : as with the hydrogen ppm box after entering the last data item DO NOT PRESS ENTERIf the cursor moves on to the next line the program will assume the last data point is a 0. The cancel button allows one to exit this section leaving all the boxes unchanged.To register all the data you need to leave this section using the OK button. The OK button is disabled until all the boxes have been correctly filled out. After entering the last data item into the oxygen or hydrogen PPM box click the cursor back up to the date. This should reenable the OK button. If it does not then click back to one of the data input boxes and then back to the date or time. The program will issue a warning if the variation in the enrichment estimates exceeds 2% of the mean for that particular isotope. This is an arbitrary limit that has been often used to screen data. The program will continue whether the variance in the data exceeds this limit or not. This could also indicate that the cursor was advanced and the program has inserted a 0 into the enrichments for one of the isotopes. If there is no background information for the individual animal then it is necessary to enter data for another individual, or some assumed background based on local water supply or the international standards. In these cases enter a dummy date and time for time the background samples were collected.4.5 SET OPTIONS The set options button allows the user to change several parameters that are used in the equations and in conversion of CO2 production to energy expenditure. The FQ or food quotient reflects the food intake of the animals in question. It is assumed that animals in energy balance will oxidise their food intake and thus conversion of CO2 to energy expenditure is performed using the FQ value. The default setting is 0.85 which minimises error in the conversion when the real FQ is unknown.The other two parameters are used to evaluate CO2 production in two pool models where the group mean dilution space ratio is compared with a population dilution space ratio and either combined with it or substituted for it depending